Bacterial signal transduction is predominated by two-component systems. These systems consist of two proteins, an autophosphorylating histidine kinase and a response regulator, which is activated by phosphorylation at an aspartate residue in a Mg2+ dependent reaction. Because of their crucial role for the survival of bacteria and lower eukaryotes and its high homology, two component systems are attractive as potential new targets for antimicrobials. In addition, these systems control the expression of virulence and drug resistance factors in several pathogenic organisms. Inhibition of the two component pathway may present an opportunity to depress resistance by targeting multiple proteins with a single inhibitor. When phosphorylated, the receiver domain ("switch" component of the resonse regulator) modulates the activity of its cognate output domain, often a transcriptional activation domain. No structure has been obtained for the phosphorylated form of either an isolated receiver domain or an intact response regulator due to the short half-life of the phospho-aspartate linkage. A long- term goal of this laboratory is to elucidate the mechanism of activation of response regulators using the transcriptional activator NtrC (nitrogen regulatory protein C) as model system. NtrC consists of three domains, the N-terminal receiver domain, the transcriptional activation domain and the DNA-binding domain. First, the structure of the transiently phosphorylated receiver domain will be determined by NMR. The main tricks used are (a) creating a steady state using large excess of phosphodonor and (b) adding multiple three dimensional data sets taken on multiple NMR samples. Second, the mechanism of activation triggered by phosphorylation will be characterized by NMR relaxation experiments and amino acid substitutions that uncouple phosphorylation and activation. The active site structure will be probed by heterologous metal ion replacement. Third, the signal cascade from the receiver domain to the transcriptional activation domain will be investigated. This problem is challenging because of the size of the full-length protein (104 kDa). Methods for segmental isotopic labeling using the splicing enzymes inteins will be combined with recently developed NMR techniques such as TROSY and dipolar couplings in liquid crystalline medium.